Mindfulness-based randomized controlled trials led to brain structural changes: an anatomical likelihood meta-analysis

Mindfulness has become increasingly popular and the practice presents in many different forms. Research has been growing extensively with benefits shown across various outcomes. However, there is a lack of consensus over the efficacy of randomized controlled mindfulness interventions, both traditional and mind–body formats. This study aimed to investigate the structural brain changes in mindfulness-based interventions through a meta-analysis. Scopus, PubMed, Web of Science, and PsycINFO were searched up to April 2023. 11 studies (n = 581) assessing whole-brain voxel-based grey matter or cortical thickness changes after a mindfulness RCT were included. Anatomical likelihood estimation was used to carry out voxel-based meta-analysis with leave-one-out sensitivity analysis and behavioural analysis as follow-ups. One significant cluster (p < 0.001, Z = 4.76, cluster size = 632 mm3) emerged in the right insula and precentral gyrus region (MNI = 48, 10, 4) for structural volume increases in intervention group compared to controls. Behavioural analysis revealed that the cluster was associated with mental processes of attention and somesthesis (pain). Mindfulness interventions have the ability to affect neural plasticity in areas associated with better pain modulation and increased sustained attention. This further cements the long-term benefits and neuropsychological basis of mindfulness-based interventions.


Methodology Study selection
Search strategy A systematic literature search was carried out in four databases, Scopus, PubMed, Web of Science, and APA PsycINFO from the earliest studies up till April 2023.This is to ensure that both the psychological and neuroscience aspects of the research topic are covered as well as any inter-disciplinary research.The keywords used for the search in both the title and abstract were: "voxel-based" or "morphometr*" or "voxelwise" or "VBM" or "brain structur*" or "structur* change*" or "structural MRI" or "structural scan*" or "gray matter" or "grey matter", together with "Meditat*" or "Mindful*" or "mind-body" or "mind body" or "yoga", and lastly with "RCT" or "intervention" or "trial".
All records that surfaced were imported into Covidence 38 which helped to flag studies that were duplicated.All the duplicated articles were checked manually before being removed from the review.Following this, the author did an abstract screen to remove articles that were clearly irrelevant to the research question.If the study was relevant or the relevance was uncertain, and it appears to have met the following eligibility criteria, the fulltext article was retrieved.Additionally, the studies included in systematic reviews and meta-analysis that were relevant to our topic was checked to ensure that we did not miss out any studies.Then, a full-text screen was conducted to ensure that all eligibility criteria were met.In addition, all the references of the papers selected in the first round of full-text review were included in the meta-analysis.

Eligibility criteria
Included studies must be RCTs with either a random or quasi-random allocation, with a treatment and a control group.Control groups could be active or passive, with some studies including both types of control groups within the study design.Only RCTs were included to conduct a more rigorous meta-analysis of mindfulnessrelated structural changes and control for individual differences that could surface when comparing expert practitioners to those without mindfulness experience in cross-sectional studies.The outcome measure must be a voxel-based or vertex-based comparison of whole-brain GM.Thus, only studies of the GM volume and cortical thickness conducted with structural MRI were included.Regions-of-interest analysis could introduce biases in terms of different methods of choosing and including the brain regions to focus on.Study populations were not restricted to healthy subjects and clinical populations were included as well, regardless of gender and race.In addition, all interventions that meet the definition of mindfulness as a form of present-moment awareness were included since the aim of the study is to surface brain structures related to mindfulness practice.Thus, both the traditional forms of interventions that examined solely the effect of mindfulness and holistic mind-body forms of mindfulness were included.
Studies with less than 10 subjects were excluded due to the low validity of the study.In addition, studies will be excluded when the required information cannot be extracted or obtained from the corresponding authors.Studies that re-analysed previously published data, protocol papers, and abstract-only papers were not included.Studies with null findings were excluded as they do not provide spatial coordinates that were necessary for the analysis method used in this study.To provide more stringent findings, studies that do not employ correction for multiple comparisons or cluster-level family-wise error correction in determining statistically significant clusters were excluded.

Meta-analysis
The preferred reporting items for systematic reviews and meta-analysis (PRISMA) 39 and the following neuroimaging meta-analysis guidelines 40,41 were followed in this study.

Data extraction
Study characteristics including the author, year of publication and country of study, subject characteristics of age, gender, education, and health or clinical status, and intervention details such as the type of mindfulness practice, length of intervention, type of randomization and control group, and sample size were extracted from the study articles.Statistical data of the analysis design, the cluster-based statistical thresholds used to determine which voxels were statistically significant, and the software, which can be Statistical Parametric Mapping, FMRIB Software Library or other packages, and stereotactic space, which can be Montreal Neurological Institute (MNI), raw Talairach, or MNI converted to Talairach using Brett transform, were also noted.Lastly, the significant brain regions with their peak coordinates, and the direction of change, which can increase or decrease for the intervention group in comparison to the control group were extracted.

Statistical analysis
Voxel-based meta-analysis were carried out with Anatomical Likelihood Estimation (ALE).ALE is derived from activation likelihood estimation principles and detects convergence among the significant coordinates reported across studies that are above chance-levels.ALE was carried out using BrainMap's GingerALE v3.0.2.Studies that reported coordinates in Talairach space were transformed to MNI space using the Lancaster transform icbm2tal provided in GingerALE.First, ALE uses a Gaussian function to model the coordinates of the studies that were included by accommodating the spatial uncertainty of significant coordinates that could be caused by differences in the neuroanatomy or by using different normalization techniques and brain templates, and by taking into account the sample size of each study.Second, each study has a whole-brain map constructed, where each voxel is assigned a number that corresponds to the probability that a difference in volume between the treatment and control groups occurs within the voxel.These maps from all the studies were then combined and yielded the ALE image where the likelihood of a particular voxel having difference in volumes found minimally for one study was represented in ALE values.Then, the statistical significance of these ALE values were analyzed with a clusterlevel family-wise error (FWE) correction threshold of p < 0.05 with 1000 permutations.The cluster-level FWE correction is known to be the most appropriate method for inferring the statistical significance of ALE analyses 42 .
Following which, the Behavioural Analysis Plugin 3.1 for Mango 4.1 was used to access the behavioural profiles and mental processes that are associated with the identified significant ALE clusters.Based on the functional metadata from the BrainMap Database, the significant ALE clusters were compared against this map to generate the behavioural domains that could fall under Interoception, Cognition, Action, Emotion, and Perception, and is further divided into 60 sub-domains.The significant ALE clusters was transformed to the Talairach space using the transform tool MNI-to-Tal provided in Mango.The region of interest of the significant ALE cluster was selected and the proportion of behavioural domains found within this cluster was compared to the proportions of the behavioural domains across the whole functional database.A behavioural domain profile of the significant cluster was thus generated where Z-scores above 3.0 were considered significant.Sensitivity analysis will be conducted using a leave-one-out method to test the replicability of the results.

Study search and characteristics
In the first round of search, a total of 254 articles were retrieved from the selected databases (PsycINFO = 44, Scopus = 88, Web of Science = 64, PubMed = 58).Duplicates were surfaced by Covidence and manually checked before deleting, which resulted in 109 studies to be screened.Following the eligibility criteria, 48 studies were found to be irrelevant during the title and abstract screening stage.Thus, 61 full-text articles were closely assessed for eligibility, leading to 10 included studies which met all the inclusion criteria.After which, all the references of the 10 included studies were entered into the selection process but only one article met all the inclusion criteria.Thus, the final number of included studies was 11.The full selection process and reasons for exclusion can be found in the PRISMA flow chart in Fig. 1 while the characteristics of the 11 included studies can be found in Table 1.Studies that were excluded for null findings were by Wolf, et al. 43 , Kral, et al. 44 , Seminowicz, et al. 45 and Mooneyham, et al. 46 while Pickut, et al. 47 was excluded for using an uncorrected threshold.
In summary, this meta-analysis had 581 participants (N = 11 studies) from various regions such as Asia (n = 5), Europe (n = 3) and North America (n = 3).All studies reported significant intervention > control contrasts while three studies reported significant control > intervention contrasts.Roughly half of the studies (n = 5) had physical components together with the mindfulness aspect such as Yoga, TCC and Baduanjin.Majority of the studies used a passive control group (n = 6), compared to an active control group (n = 3), while two studies had both an active and passive control groups.The active control groups ranged from memory enhancement training and aerobic exercise to health education programmes.Lastly, about half the studies (n = 6) were on healthy participants while the rest were on patient populations such as those with Mild Cognitive Impairment (MCI) or chronic neuropathic pain.  1 SD was not provided and if range was provided, the range rule, whereby the SD of the sample is approximately 1/4th the range of the data, is used to estimate it. 2Authors combined the ACG that did a colouring task with the PCG because of high dropout rates. 3This was assumed from the study's methods as it was not explicitly stated. 4These demographic variables were not based on the final sample but the sample at baseline. 5Number of females in the final sample with usable MRI scans were not known. 6Intervention protocol involves multiple training modules and has been simplified here for clarity's sake.Please refer to the paper for more details. 7This study had both grey matter volume and cortical thickness data.

Risk of bias of included studies
The revised tool to assess the risk of bias in randomized trials were used in this assessment 59 .Availability of outcome data for nearly all participants randomized were considered when drop-out rates after the intervention were less than 10%.All included studies were assessed to be of low risk, as seen in Fig. 2.

Significant ALE clusters
One significant cluster, as seen in Fig. 3, emerged from the intervention > control ALE contrast and covered the right insula and precentral gyrus in the Brodmann area 13 (BA13) and 44 (BA44) respectively.The cluster size was 632 mm 3 with one peak voxel MNI coordinates at 48, 10, 4. The ALE value for this cluster was 0.017 (p < 0.001, Z = 4.76) with two contributing experiments by Santarnecchi, et al. 56 and Krause-Sorio, et al. 53 .There were no significant clusters from the intervention < control ALE contrast.Moreover, since both GM and cortical thickness measures were included, we re-conducted the ALE meta-analysis without the cortical thickness measures to see if that affected the results and no clusters remained significant.

Sensitivity and behavioural analysis
Using the leave-one-out method, the stability of the findings were tested.The region of interest that emerged above remains significant in all except two of the repetitions, when the studies that contributed to the clusters were excluded.These occurred when we left out the two studies that contributed to the significant cluster above.Behavioural analysis of the significant ALE cluster above indicated that the cognitive subdomain of attention (Z = 3.77) and the perception subdomains of somesthesis, in particular pain (Z = 3.01) were the mental processes associated in the region.

Discussion
Across the intervention groups, there was a significant structural increase in the right insula and precentral gyrus region.Using behavioural analysis, this cluster was found to be associated with the cognitive process of attention and the perception process of pain-sensing.This region of interest remained significant in all but two of the repetitions, occurring when the studies contributing to the significant clusters were removed 53,56 .This signifies that the ALE cluster was due to a small set of studies.However, this brain region was consistently activated even though the type of mindfulness practice was varied, with one using yoga as the intervention while the other used MBSR.Interestingly, one sample was healthy while the other used participants who were at risk for Alzheimer's disease.
The insula is often implicated in both sensorimotor and emotional processing, especially during interoceptive experiences 60 .The insula is also involved in top-down control of pain expression and transmission 61 which could explain findings of improved pain symptoms amongst those suffering from chronic pain following mindfulness interventions 62,63 .The posterior insula, where Brodmann area 13 is located, is crucial in processes like emotion regulation and attentional control 64 .The neural plasticity in this brain region with engagements in mindfulness practice could be the mediating factor for the well-being outcomes often observed.The right insula in particular has shown more involvement in self-related processing than the left due to specific neural features 65 .With mindfulness practice largely focused on an awareness of the self, it could explain this observation of a unilateral structural change in the insula.
The precentral gyrus, similar to the insula is often activated during interoceptive attention 66 .The right precentral gyrus especially is activated during awareness of the self 67 and the right side of prefrontal activation is often observed during sustained attention tasks 68 .Overall, it seems that the mechanisms underlying mindfulness-based interventions stem from improvements in attention, especially attention inward to the self, and pain processing.Given the research surrounding the overlap of brain regions activated during both emotional and physical pain 69 , an increase in the structural volume of the right insula and precentral gyrus could contribute to the benefits of mindfulness practice as a therapeutic tool.
In the most recent meta-analysis on GM changes relating to mindfulness meditation, only structural change in the right anterior ventral insula was found to be consistent across studies 29 .The study excluded any mindfulness interventions with physical components, such as yoga and the popular MBSR programmes and the brain region found to be significant was similar but more in the anterior region, compared to the findings of this study which saw more structural changes in the posterior insula.The selection of included studies was also mentioned as a weakness since most were cross-sectional.The studies compared structural volume differences in the brains of meditators and non-meditators at a single timepoint or looked at the association of meditation experience with structural brain volume in meditators only.Thus, the stringent inclusion criteria set in this meta-analysis helped to address issues relating to experimental biases in previous meta-analyses.The insula was also consistently activated across fMRI studies during meditation tasks among participants with no experience in mindfulness 35 .It seems that the benefits of the intervention could be enduring given the structural change observed in this same region.
Another comprehensive and widely cited meta-analysis on morphometric changes associated with mindfulness practice mainly looked at significant differences between long-term practitioners and participants with no prior experience 26 .Only five studies used a mindfulness intervention on participants with no prior experience.Thus, there were more significant structural differences in brain regions observed in the anterior-and midcingulate cortex, middle frontal gyrus, anterior precuneus, fusiform gyrus, orbitofrontal cortex, inferior temporal gyrus, somatomotor cortices, and anterior insula.These additional brain regions could be attributed to either longer-term effects of consistent mindfulness practice, or biases arising from the cross-sectional design of the study.In contrast, the findings from our meta-analysis shows that even short-term mindfulness practice can effect structural changes in the brain.The two studies that contributed to the significant cluster found had mindfulness intervention durations between 8 to 12 weeks with 1 session per week.8 weeks of mindfulness intervention was also the most common intervention duration used and could be the minimum period for mindfulness interventions to reflect effectiveness in terms of structural brain changes.This provides more convincing support for the benefits of mindfulness practice and allows us to allude the improvements in awareness and pain processing to the practice itself.Moreover, it seems that the benefits do not require years of mindfulness practice before appearing but we should take caution in interpreting the longevity of these changes since post-intervention follow-ups are usually not carried out in the studies.
The number of studies included in this meta-analysis did not meet the recommended good practice amount of 17 by 40 .However, we believe that it is crucial to explore what the current landscape of high-quality RCTs provides in terms of the evidence of the benefits of mindfulness intervention as interest in the area grows.This also surfaces multiple future research directions.With an increased sample size of included studies, future subgroup analysis could compare the effects of an active versus passive control group, or the effects of including a physical component to the mindfulness intervention, potentially isolating the unique mechanisms of the various components of mindfulness practices.Additionally, differential effects on a healthy versus patient population could also be explored.It is interesting that the patient populations in this meta-analysis have impairments in www.nature.com/scientificreports/areas such as pain-processing and cognitive deficits which overlaps with the mental processes associated with the significant brain regions found.There is a possibility that the effectiveness of mindfulness interventions only pertains to clinical populations where deficits are seen in attention and pain-processing.However, future studies are needed to confirm that.Unfortunately, it was also not possible to verify the effect of potential confounding variables such as age, gender, and length of intervention using meta-regressions with the ALE method, and the exclusion of studies without significant results also causes a selection bias.An important study that was excluded because of nonsignificant results was a large-scale and rigorously controlled study by Kral et al. 44 .It is possible that they did not detect significant GM changes due to their stringent exclusion criteria which included anyone with expertise in nutrition, music or physical activity.Additionally, participants had to undergo various other assessments as part of a larger multi-project study with each visit taking 2-4 h which could introduce other variabilities.These are all potential areas that future studies should take into account.
In conclusion, this meta-analysis has found evidence for structural brain changes following mindfulness interventions.These interventions, both traditional and mind-body formats, have the ability to affect neural plasticity in the brain regions associated with pain modulation and sustained attention.During the mindfulness intervention, the repeated practice of engaging one's attention and awareness to the self repeatedly activates these regions of the brain.Over time, neural plasticity could lead to an increase in the volume of these regions.This further cements the long-term benefits and neuropsychological basis of mindfulness-based interventions.

Figure 1 .
Figure 1.PRISMA flow chart of selection process.Selection process of included studies for this meta-analysis according to PRISMA guidelines.

Figure 2 .
Figure 2. Risk of bias assessment.Results of the risk of bias assessment for all included studies.

Figure 3 .
Figure 3. Results of the ALE meta-analysis.The highlighted cluster represent significant convergence of structural increase in mindfulness intervention participants compared to controls.The cluster is depicted on an MNI standard brain template and the colour indicates the ALE values.L = left; R = right, A = anterior, P = posterior.

Table 1 .
Characteristics of included studies.SD standard deviation, IG intervention group, PCG passive control group, ACG active control group, TCC Tai Chi Chuan, TAU treatment as usual, aMCI amnestic mild cognitive impairment, AD Alzheimer's disease, MBSR mindfulness-based stress reduction, US United States.